Natural restriction enzymes are now essential for gene engineering and many types of restriction enzymes from microbes, plants, or animals are commercially available. Various kinds of these enzymes are known, for example, EcoR I which is isolated from Escherichia coli. They are very expensive, must be handled under conditions of low temperature and require storage in a refrigerator or freezer at below −20° C. In addition, it is impossible to design the cleaving point artificially because they are natural proteins.
So called “tailor-made” polynucleotide cleavage agents which can cleave DNA or RNA selectively at a desired position, and which can be stored at room temperature are therefore required.
Many types of natural DNA cleavage agents are known to work as strong anti-tumor or anti-cancer medicines. Among them, calicheamicin is a well respected potent anti-tumor medication. The origin of its DNA cleavage activity has been proposed as being from a biradical intermediate that is produced by the degradation process from an unstable ene-diyne group to a stable aromatic ring.
Extensive studies have been made to develop an artificial restriction enzyme which can cleave a DNA sequence selectively. Recently, simple anthracene or anthraquinone derivatives have been reported as potent DNA cleavage agents: Schuster and co-workers reported that AQC caused GG selective cleavage of duplex DNA (J. Am. Chem. Soc. 1996, 118, 8747), and Kumar succeeded in cleaving DNA by simple anthracene derivatives (Tetrahedron, 2000, 56, 7027). Toshima developed a novel quinoxaline-carbohydrate hybrid as a GG-selective DNA cleaving agent with DNA cleavage and binding abilities which are dependent on the structure of the sugar moiety (Angew. Chem., Int. Ed. Engl. 1997, 36, 2748; Bioorganic & Medicinal Chem. Lett. 2000, 10, 2163; Chem. Commun., 212 (2002)). Artificial restriction enzymes by photo-irradiation which can be practically used have not been reported.
During the course of the study on the synthesis of optically active gem-difluoro-cyclopropane derivatives, we noted an interesting fact: the preparation of 9-anthracenecarboxylic acid ester of 1,3-bishydroxymethyl-2,2-difluoro-cyclopropane has to be performed under darkened conditions. Also, some papers on gem-difluoro-cyclopropane ring disclosed that an unidentified ring opening product was formed instead of the desired diester when the reaction was carried out under visible light irradiation conditions (Chem. Lett. 1998, 903; Tetrahedron Lett. 1999, 40, 5739), and a gem-difluoro-cyclopropane ring was easily decomposed and caused a ring opening reaction via a radical intermediate (Dolbier, Jr. et al., J. Org. Chem. 1999, 64, 540).
These findings stimulated to investigate the possibility of gem-difluoro-cyclopropane derivatives of 9-anthracene carboxylic acid as novel DNA cleavage agents. It was anticipated that strong DNA cleavage activity could be obtained by the synergetic effect of the difluoro-cyclopropane group with the anthracene carboxylic group. However, it was confirmed that there was no significant DNA cleavage activity for such 9-anthracene carboxylic diester of 1,3-bishydroxymethyl-2,2-difluoro-cyclopropane (The 25th Japanese Symposium on Fluorine Chemistry, Nov. 18, 2001, p. 54–55). Extensive studies have been made to develop strong DNA cleavage agents that can cleave a DNA sequence selectively when switched on by photo-irradiation and such compounds could serve as an anti-tumor medication.